Hepatitis B virus causes chronic infections in approximately 350 million people worldwide. All are at significantly increased risk of developing hepatocellular carcinoma. Although virus replication can be blocked by therapy with nucleoside analogs, infected hepatocytes are not cured because covalently closed circular DNA (cccDNA), the template for transcription of viral RNAs, persists in infected cells. So far, therapeutic strategies to prevent cccDNA formation or functionally inactivate or even destroy cccDNA within infected hepatocytes is lacking. From a basic as well as translational science point of view, two major gaps in knowledge need to be filled in order to develop novel curative therapeutic approaches targeting cccDNA. First, information is required about the stability of cccDNA in dividing cells. Is cccDNA lost during cell division? Can cccDNA loss be induced or enhanced through environmental cues, such as those induced by certain cytokines? Secondly, information is required about the nature of cellular enzymes required for cccDNA formation from the relaxed circular (rc) DNA genome. Is cccDNA formed by a DNA repair mechanism similar to NER (nucleotide excision repair) or GGR (global genomic repair) pathways used by cells to maintain genome integrity? What are the enzymes required for this process? The purpose of this grant application is to address these questions with an experimental approach that relies on a novel assay system for HBV infections in cell culture recently developed in our laboratory. It combines the NTCP (sodium taurocholate co-transporting polypeptide) dependent HepG2 assay for HBV infections together with the highly efficient CRISPR/Cas9 system for gene knockout, into a single platform for identification of genes required for HBV infection. Specifically, we will investigate i) the fate of cccDNA during cell division, ii) the role of the previously described cellular factor TDP2 (tyrosyl DNA phosphodiesterase-2) in cccDNA formation and iii) the DNA repair mechanism required for the formation of cccDNA from rcDNA. A better understanding of the fate of cccDNA during cell division is of critical importance for antiviral therapy because the failure of nucleotide analogue therapy to cure chronic hepatitis B (CHB) is believed to be a consequence of cccDNA stability. The proposed experiments will provide information that could pave the way for novel approaches that will enhance the loss of cccDNA and hence increase the chances for a cure of CHB. Knowledge about the cellular enzymes required for the repair of rcDNA and formation of cccDNA will identify new targets for antiviral therapies that do not carry the risk for the development of resistance, as is observed with conventional antivirals. In addition to the significance for public health, this application has significance for the field of virology in general because it could fill important gaps in knowledge concerning the life cycle of hepadnaviruses.